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Department of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, the State University of New York, Buffalo, New York 14214
Address all correspondence and requests for reprints to: Dr. S. Laychock, 102 Farber Hall, the State University of New York at Buffalo, School of Medicine, Buffalo, New York 14214. E-mail: laychock{at}acsu.buffalo.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-methene-oxidizing,
hydroxylating), EC 1.14.99.3], also known as heat shock protein
(hsp)32, is a membrane-bound enzyme responsible for catalyzing heme
degradation. HO uses dioxygen and nicotinamide-adenine dinucleotide
phosphate as cofactors, with the resulting products of the reaction
being carbon monoxide, iron, and biliverdin (1). Biliverdin is
converted to bilirubin by a ubiquitous cytosolic enzyme biliverdin
reductase (2). HO exists in two forms as products of distinct genes:
HO-2 (36 kDa) is constitutive, and HO-1 (32 kDa) is inducible. Both
HO-2 and an inducible HO-1 have been identified in rat pancreatic
islets (3, 4, 5), as well as other tissues (6). HO-1 expression increases
in response to heme and stressors such as UV radiation and oxidative
stress, as well as endotoxin, hormones, and heavy metals (6). HO-1
induction may protect cells by reducing heme levels that catalyze
oxygen radical reactions and elevating bilirubin, which has antioxidant
properties (1). Bilirubin inhibits autoxidation or
peroxyl-radical-induced oxidation of unsaturated fatty acids,
apparently through peroxyl radical-trapping antioxidant abilities (7, 8). In addition, bilirubin scavenges and quenches toxic singlet oxygen
(9). The expression of HO-1 is regulated by the family of AP-1
transcription factors, among others, and the expression and DNA binding
activity of c-Fos and c-Jun are stimulated by prooxidants such as heavy
metals, hydrogen peroxide, and UV-irradiation (10, 11). Pancreatic islets respond to stress through the induction and activation of several stress-activated proteins. Interleukin-1ß (IL-1ß) induces an inflammatory response in pancreatic islets, characterized by increases in inducible nitric oxide synthase (iNOS) levels and increased nitric oxide (NO)/nitrite levels (12, 13, 14, 15). IL-1ß and heat shock increase expression of hsp70 (16, 17), as well as HO-1 (3, 18). A protective effect of heat shock on islet cells may be associated with reduced lysis from NO, reactive oxygen intermediates, and streptozotocin (17); but the response is nonspecific because many hsp respond to this stimulus. On the other hand, liposomal delivery of hsp70 into islet cells protected the cells from IL-1ß effects on insulin secretion (19), suggesting that heightened levels of specific hsp can protect ß-cells from inhibitory effects of the cytokine. Hemin, which increases HO-1, has also been found to partly counteract the IL-1ß inhibition of insulin release and to protect against IL-1ß-induced inhibition of aconitase activity and glucose oxidation (18), perhaps through antioxidant mechanisms. However, hemin has also been reported to increase insulin and glucagon secretion from normal rat islets (4). The present study investigates the potential for a heavy metal to induce the synthesis of HO-1 and affect pancreatic ß-cell responses to IL-1ß.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue isolation/culture
Isolated pancreatic islets were prepared from male rats and were
cultured for 20 h in CMRL-1066 medium containing 5.5
mM glucose, as described previously (20). Other agents
present during culture are specified in the text. All animal procedures
were approved by the Institutional Animal Care and Use Committee. When
sodium nitroprusside (SNP) was used to generate nitric oxide, cyanide
ions were inactivated by rhodanese (1.3 U) and sodium thiosulfate (25
µM) (21).
ßHC9 insulinoma cells, from an established line derived from hyperplastic pancreatic islets of transgenic mice harboring SV40 large T-antigen in ß-cells (22), were a gift from Dr. D. Hanahan. The cells were cultured in complete DMEM medium, deficient in pyruvate and containing 10% bovine FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml), at 5% CO2-95% air, 35 C.
Glucose oxidation
Glucose oxidation was determined by quantitation of picomoles of
glucose oxidized to 14CO2, based upon the
specific activity of [U-14C]glucose (17 mM),
as described previously (23).
Insulin release
Isolated islets (10/sample) were cultured 20 h in CMRL-1066
medium in the absence or presence of IL-1ß (1 ng/ml) and other
agents, as indicated in the text. Cultured islets were subsequently
washed to remove culture medium, serum, and experimental agents. Washed
islets were preincubated for 1 h in Krebs Ringer bicarbonate (KRB)
buffer (pH 7.4), containing 5.5 mM glucose, 0.01% BSA, and
HEPES (16 mM) but lacking IL-1ß or the other agents
present during the overnight culture, as described previously (23).
After preincubation, the islets were placed in fresh KRB buffer, and an
aliquot was removed to determine zero-time insulin levels. Islets were
incubated at 5.5 or 17 mM glucose for 60 min, after which
an aliquot of the incubation buffer was removed to determine insulin
release. Insulin was quantitated by RIA, and zero-time insulin levels
were subtracted from 60 min values. Insulin in culture medium was also
quantitated after the overnight culture. Total islet insulin content is
the amount of insulin extracted from islets, using ethanol (70%) in
HCl (1 N), plus the insulin released during static incubation.
Nitrite measurements
NO synthesis was estimated by the accumulation of total nitrate
and nitrite in culture medium of islets after an 18-h culture, as
described previously (23).
Western blot analysis
Isolated islets were cultured, as described above, and sonicated
in deionized water (0.1 ml) containing leupeptin (5 µg/ml), aprotinin
(76 µg/ml), and pepstatin (1 µg/ml), at 4 C. Islet protein levels
were determined by Bio-Rad protein assay using BSA as standard. The
remaining islet protein was precipitated using a 10x volume of
ice-cold acetone and microcentrifugation at 14,000 rpm for 5 min;
protein recovery was 94% using BSA as standard. The protein was
resuspended in SDS sample mix (0.062 M Tris-HCl, 1%
ß-mercaptoethanol, and 2% SDS) containing protease inhibitors, as
specified above, and boiled for 5 min. Proteins were separated by
SDS/15% PAGE (24). Equal amounts of protein per sample were used in
each experiment. Proteins were electrophoretically transferred to
nitrocellulose membranes and reacted with rabbit antirat HO-1 (1:1250
dilution) or antimouse hsp70 (1:500 dilution), followed by horseradish
peroxidase-conjugated goat antirabbit or antimouse IgG. Detection of
HO-1 was by the enhanced chemiluminescence method and Kodak AR-10 X-ray
film exposure. Each experimental determination was repeated at
least two or three times. Densitometric analysis of each band on
Western blot was analyzed by Molecular Analyst software (Bio-Rad
Laboratories, Hercules, CA). The data for each experiment were
normalized to control values by subtracting the image density of
control HO-1 levels from each experimental sample for quantitative
analysis. Values for densitometric analysis are expressed as relative
density units (RDU).
Heme oxygenase (HO) activity
Total HO activity was quantitated by the generation of bilirubin
from heme in cell homogenates, essentially as described previously
(25). Treated and untreated ßHC9 cells were incubated for 18 h;
and then the cells were collected, washed with PBS, and suspended in
MgCl2 (2 mM) phosphate (100 mM)
buffer (pH 7.4). The cells were frozen and thawed three times,
sonicated, and centrifuged at 5000 rpm for 5 min in a microfuge at 4 C.
An aliquot of the supernatant (0.3–0.4 mg protein) was added to the
NADPH-generating system (0.4 ml) containing NADPH (0.8 mM),
glucose-6-phosphate (2 mM), glucose-6-phosphate
dehydrogenase (0.2 U), potassium phosphate buffer (100 mM,
pH 7.4), hemin (10 µM), and 2 mg protein from rat liver
(35,000 rpm supernatant fraction) as a source of biliverdin reductase,
and allowed to incubate for 2 h at 37 C in amber tubes. The
reaction was stopped, and bilirubin was extracted by addition of 0.8 ml
chloroform; the aqueous layer was extracted twice with additional
chloroform. The bilirubin extract was dried and resuspended in 0.5 ml
chloroform for determination of bilirubin by the difference in
absorption at 464 nm and 530 nm. Readings for samples, prepared in the
absence of cell homogenate (blank), were subtracted from all other
values prepared in duplicate. HO activity is expressed as pmol
bilirubin produced/mg protein·2 h.
Determination of zinc-protoporphyrin IX (ZnPP) uptake by
cells
ßHC9 cells were cultured 20 h with ZnPP (10
µM). Then, the cells were collected, washed with PBS
three times, and resuspended in formic acid (88%), as described
previously (25). The spectrophotometric absorbance was read at 407 nm,
and cell extract absorbance was compared with ZnPP standard to
quantitate uptake.
Statistical analysis
The data are presented as the mean ± SE and
were analyzed by one-way ANOVA combined with the post hoc
Student/Newman-Keuls multiple-comparison test; P <
0.05 was accepted as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Culture of rat
islets has been previously reported to increase HO expression (5),
perhaps through accumulation of glucagon (6) or factors present in the
culture medium/serum. Islet culture with IL-1ß also induced a small
increase in HO-1 levels, which were slightly but consistently above
control values (Figs. 1A and 2). The
presence of cobalt chloride (CoCl2) during islet
culture induced a high level of expression of HO-1, which was
concentration-dependent from 1–100 µM (Figs. 1B and 2).
In contrast, the presence of similar concentrations of nickel chloride
(NiCl2) in the islet culture did not induce comparable
changes in HO-1 levels, compared with CoCl2 (Fig. 1A).
CoCl2, in combination with IL-1ß, also enhanced HO-1
levels above those observed with the cytokine alone (Figs. 1A and 2),
whereas NiCl2 did not affect IL-1ß-induced HO-1 levels
(data not shown). However, islet HO-1 levels in the presence of IL-1ß
and CoCl2 (10 µM) were significantly lower
than in the presence of CoCl2 alone, perhaps because of
cytokine effects on protein synthesis. A time-course analysis showed
that CoCl2 induced an increase in islet HO-1 levels within
8 h, and IL-1ß induced an increase after 18 h of culture
(Fig. 3, A and B). When both
CoCl2 and IL-1ß were present together in islet culture,
HO-1 levels were increased within 4 h, and much higher levels were
observed within 8 h, compared with islets treated with IL-1ß
alone (Fig. 3C). In comparison with HO-1, islet hsp70 levels did not
seem to be affected during islet culture with CoCl2 or with
the concentration of IL-1ß used in this study (Fig. 3, A and B).
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, A and B). The maximum concentration of
SNP inducing HO-1 expression was 100 µM, and a higher
concentration (250 µM) seemed to be less effective,
perhaps because of toxic effects on the cells. To determine whether NO
production mediated the effect of IL-1ß on HO-1 expression, an
inhibitor of nitric oxide synthase, NMMA, was included during the islet
culture with IL-1ß. Western blot analysis revealed that, whereas
IL-1ß (1 ng/ml) increased islet HO-1 levels above control, islets
cultured with NMMA (1 mM) in the presence of IL-1ß had
significantly lower HO-1 levels, which were similar to levels in islets
treated with NMMA alone (Fig. 2
).
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). The increase in basal
insulin release in islets cultured with 10 µM
CoCl2 was metabolically regulated, because release was
completely inhibited in cells at 4 C and was also calcium-dependent in
studies performed in calcium-free KRB buffer in the presence of EGTA
(10 µM) (data not shown). NiCl2 (10
µM) did not increase basal insulin release (Fig. 5A).
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IL-1ß characteristically inhibited glucose-stimulated insulin release
from islets incubated with the cytokine for 20 h (Fig. 5B).
However, when CoCl2 was included in the 20-h culture with
IL-1ß, then subsequent glucose-stimulated insulin release responses
were higher than observed in islets exposed only to IL-1ß and glucose
(Fig. 5B). The protective effect of CoCl2 was
concentration-dependent, with as little as 1 µM
CoCl2 affording protection of the islets from the
inhibitory effect of IL-1ß on glucose-stimulated insulin release.
Culture of islets with CoCl2 (10 µM) alone
did not affect glucose-stimulated insulin release (Fig. 5B). Culture of
islets with NiCl2 (10 µM), in the presence or
absence of IL-1ß, did not significantly affect glucose-stimulated
insulin release, compared with control values (Fig. 5B).
Cobalt effects on glucose metabolism
Culture of islets with IL-1ß reduced glucose oxidation by about
40% (Fig. 6). Culture of islets with
CoCl2 (10 µM) alone did not affect glucose
oxidation at 17 mM glucose (Fig. 6) or 5.5 mM
glucose (data not shown). However, the presence of CoCl2
prevented the reduction in glucose oxidation observed with IL-1ß
(Fig. 6). The presence of NiCl2 (10 µM) did
not affect glucose oxidation, and NiCl2 did not protect the
islets from the inhibitory effects of IL-1ß (Fig. 6).
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). ZnPP (10 µM) did not
affect basal insulin release at 5.5 mM glucose in either
control islets, IL-1ß-treated islets, or IL-1ß plus
CoCl2 (10 µm)-treated islets (data not shown). Glucose
(17 mM)-stimulated insulin release in IL-1ß-treated
islets was significantly inhibited and was not affected by the presence
of ZnPP (10 µM) (Fig. 7B). However, the inhibitory
response to IL-1ß was completely reversed by the presence of
CoCl2 during culture (Fig. 7B). In contrast, ZnPP (10–100
µM) reversed the protective effect of CoCl2
in IL-1ß-treated islets on glucose-stimulated insulin release (Fig. 7B).
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ZnPP did not affect expression of HO-1 in control islets or in islets treated with CoCl2 and IL-1ß (data not shown). Studies using ßHC9 cells showed that ZnPP (10 µM) accumulated in cells (0.31 ± 0.04 nmol ZnPP/106 cells) after 20 h culture.
In contrast to the inhibitory effect of ZnPP, cobalt-protoporphyrin IX
(CoPP) is reported to stimulate HO-1 activity (6). In islets cultured
with CoPP, there was a concentration-dependent (10–100
µM) increase in HO-1 expression, which was antagonized at
the lower concentration by the presence of IL-1ß (Fig. 2). CoPP
(10–100 µM) did not affect basal insulin release in
control, IL-1ß-treated islets, or IL-1ß plus
CoCl2-treated islets (Fig. 8A). Similarly, in CoPP (10–100
µM)-treated islets, glucose-stimulated insulin release
was not affected (Fig. 8B). However, when CoPP was included during
culture of islets with IL-1ß, then glucose-stimulated insulin release
was maintained at control levels observed in the absence of cytokine
(Fig. 8B). The presence of CoPP with CoCl2 and IL-1ß
during islet culture also preserved glucose-stimulated insulin
secretory responses, which were comparable with control
glucose-stimulated values in the presence or absence of
CoCl2 (Fig. 8B).
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). When ßHC9 cells were cultured
18 h in the presence of IL-1ß, however, HO activity in cell
homogenates was not increased, relative to control values (Fig. 9A). In contrast, CoCl2 and
CoPP increased HO activity, and HO activity after treatment with CoPP
and IL-1ß was significantly higher than activity in cells treated
with IL-1ß alone (Fig. 9A). ZnPP markedly inhibited HO activity in
control and CoCl2-treated cells (Fig. 9A).
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). When added directly to the
cell homogenate, ZnPP inhibited HO activity, whereas CoCl2
did not affect HO activity, compared with control (Fig. 9B).
Cobalt effects on nitrite production
Islets cultured with IL-1ß showed a marked increase in nitrite
production (Fig. 10). Although
CoCl2 (10 µM) did not affect basal levels of
nitrite, the inclusion of CoCl2 with IL-1ß during islet
culture significantly reduced islet nitrite production (Fig. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (34). In the present study, CoCl2
also protected against the effects of IL-1ß in islets, and one major
pathway associated with cobalt exposure of cells is induction of HO-1.
It has been previously reported that CoCl2 effects on cells
do not include induction of the expression of the hsp70 gene (35), and
our results confirm that changes in hsp70 do not parallel changes in
HO-1 in response to CoCl2 or IL-1ß at the concentrations
used in this study. It was previously reported that IL-1ß increased
hsp70 and HO-1 levels in isolated ß-cells after 24 h culture
(36). In the present study, hsp70 was also clearly expressed in islets,
although it was not evident that IL-1ß or CoCl2 enhanced
those levels. The high basal levels of hsp70 in the present study, as
also reported for isolated ß-cells (36), may have minimized the
changes detectable with the stimuli, but the results suggest that HO-1
responses and effects are independent of changes in ß-cell hsp70
levels. Co2+ can also antagonize calcium fluxes in cells,
but this occurs at high (millimolar) concentrations of the cation (26)
and results in inhibition, rather than stimulation, of hormone release.
Because calcium influx is a major component of glucose-stimulated
insulin release (37) and secretory responses were calcium-dependent in
the present study, it does not seem that the protective effects of
CoCl2 during IL-1ß exposure can be attributed to reduced
calcium fluxes. HO is found in pancreatic islets as both the inducible HO-1 and constitutive HO-2 enzymes (3, 4, 5). The increase in HO-1 in islets during short-term culture confirms the observation that rat islet HO increased dramatically, compared with human islet HO, during 5–8 days culture (5). A putative role for HO-2 in glucagon and insulin secretion has been proposed, especially in regard to the generation of carbon monoxide (4). In the present study, the increased basal insulin release in islets treated with CoCl2 may be related to enhanced HO-1 expression, because NiCl2 failed to mimic the HO-1 response or evoke insulin secretion in this study. Whether or not carbon monoxide mediates HO-1 and CoCl2 effects on insulin secretion, as suggested by other investigators regarding HO-2 (4), is not known.
Exposure of islets to CoCl2 induced HO-1 expression in a concentration- and time-dependent manner. Previous reports indicate that HO-1 expression is increased within a few hours after a stimulus is applied, although levels of HO-1 remain elevated for several days (6, 38). In the present study, CoCl2 increased islet HO-1 levels within 4 h, and maximum expression was observed within 8 h; a similar but augmented response was observed when IL-1ß was combined with CoCl2. The reduced expression level of HO-1 in islets treated with IL-1ß and CoCl2vs. CoCl2 alone suggests that IL-1ß has effects on protein biosynthesis. A similar inhibitory effect of IL-1ß on low-concentration CoPP-induced HO-1 expression was observed. However, a higher concentration of CoPP overcame the inhibitory effect of IL-1ß, suggesting that islets retained their protein biosynthetic capacity.
Treatment of islets with IL-1ß alone also enhanced HO-1 levels above control to a small extent, in agreement with previous reports (18, 33, 36, 39). In islets, NO seems to mediate the HO-1 response to IL-1ß, because HO-1 expression was increased in a time- and concentration-dependent manner in response to the NO-generating agent SNP, as reported for endothelial cells (25). Moreover, the inhibition of iNOS by NMMA was associated with a reduction in HO-1 expression in response to IL-1ß, supporting the hypothesis that NO mediates the IL-1ß-induced increase in HO-1. Because NO increases free intracellular heme (40), the increased HO-1 expression in response to NO may be a response to protect cells from free heme through metabolism to biliverdin and bilirubin. The question arises as to why IL-1ß-induced increases in HO-1 are not protective against the cytokine? One explanation may be that IL-1ß did not increase HO-1 levels until late after initial exposure to the cytokine, perhaps too late to exert a protective effect. A second explanation may be that although IL-1ß modestly increases HO-1 protein levels, the HO-1 activity may not parallel the change in expression. HO-1 activity is inhibited by NO and PG E2 (41, 42), both of which are induced during cell exposure to cytokines in islets and other cell types (43, 44). Thus, impaired HO-1 activity may not be sufficient to protect the ß-cell from the toxic effects of IL-1ß. The results show that CoCl2 treatment markedly reduces the time for expression of HO-1 and augments the HO-1 levels in IL-1ß-treated islets, compared with islets treated with IL-1ß alone, which may contribute to the protective response observed with CoCl2. The failure of NiCl2 to induce HO-1 in islets or to protect the secretory response from the effects of IL-1ß illustrates the specificity of the Co2+ response. Although Ni2+ is a heavy metal, it is known that tissues differ greatly in their responsivity to different metals, regarding HO-1 induction (40).
The specificity of the protective response of cells to CoCl2 was demonstrated also in the maintenance of glucose oxidation in IL-1ß-treated islets, whereas a similar concentration of NiCl2 proved ineffective. IL-1ß has been reported previously to inhibit glucose oxidation (23, 45, 46), apparently because of generation of NO. Glucose-stimulated insulin release is dependent upon ATP production and regulation of ATP-sensitive K+-channels, which regulate cell depolarization, calcium influx through voltage-dependent calcium channels, and secretion (37). NO causes ADP-ribosylation and nitrosylation of certain enzymes in the glucose-oxidative pathway and mitochondrial enzymes (13, 40), in addition to reducing glucokinase messenger RNA (mRNA) and protein levels (46). The protective actions of CoCl2 on insulin release may be mediated, in part, through preservation of glucose metabolism and energy production as a result of the inhibition of iNOS activity and reduced NO production. CoCl2-treated islets showed a significant reduction in total nitrite levels, which may be protective against the effects of IL-1ß. CoCl2 effects on nitrite production may be mediated through changes in iNOS activity, because one of the products of HO-1, carbon monoxide, inhibits iNOS by binding to its heme moiety (47, 48). HO-1 induction and increased heme metabolism may also limit iNOS activity caused by heme restriction. Because NO is a potent stimulator of soluble guanylyl cyclase, it does not seem likely that any additional cyclic GMP formed as a result of carbon monoxide stimulation of guanylyl cyclase (49, 50) contributed to insulin release.
Additional evidence for the involvement of HO-1 in the CoCl2 effect on glucose-stimulated insulin secretion in IL-1ß-treated islets was the concentration-dependent reversal of the protective effect by an inhibitor of HO-1, ZnPP (6). Because ZnPP-treatment did not affect glucose-stimulated insulin release in cells treated or not with IL-1ß, and ZnPP did not affect HO-1 expression, the antagonistic response would seem to be modulated through HO-1 activity changes. Previously, ZnPP was reported to inhibit HO-2 activity and carbon monoxide production in rat islets and to inhibit secretory responses induced by glucose and hemin, a substrate and inducer of HO (4). After overnight culture of islets with ZnPP, we failed to observe the small inhibition of glucose-stimulated insulin release that was reported after acute treatment of freshly isolated islets with ZnPP (4), although the reason for this is not evident. However, the present results confirm that hemin protects against the effects of IL-1ß on insulin release (18) and extend the observations to include ZnPP antagonism of the hemin protective response, probably caused by inhibition of HO activity.
Selectivity of the ZnPP response in the present study was demonstrated when CoPP, which increased HO-1 expression in islets, in agreement with the powerful inducer effects reported for other tissues (6), did not antagonize the CoCl2 response. On the contrary, CoPP increased the expression of HO-1 in islets and mimicked the islet response to CoCl2, in terms of protecting the cells from the inhibitory effects of IL-1ß. The ability of CoCl2 to induce HO-1 in rat liver requires conversion of Co2+ into CoPP (51); however, it is not known whether this conversion is responsible for CoCl2 HO-1-inducing activity in islets. Although certain of the metal-protoporphyrin inhibitors are selective for HO-1, they can also inhibit iNOS (52). However, if ZnPP inhibited iNOS in islets, then it would be expected that the islets would be protected against the effects of IL-1ß, and this did not occur. However, a inhibitory effect of CoPP on iNOS cannot be ruled out, and the interference by CoPP in the Greiss reaction for nitrite determination prevented assessment of this possibility. It is also possible that Co2+ or CoPP interfere with NO actions in cells or modify IL-1ß responses. ZnPP has been reported to reduce IL-1ß responses (53). However, the concentration of ZnPP chosen in the present study did not significantly affect IL-1ß responses in the absence of inducers of HO-1, suggesting the agent did not directly affect the cytokine response.
One of the inconsistencies in our results, regarding the hypothesis that HO-1 induction mediates protective responses against IL-1ß in islets, was the observation that HO-1 levels at a low concentration of CoPP in IL-1ß-treated islets was similar to levels in islets treated with IL-1ß alone, and yet insulin release responses were protected. Investigation of the HO activity in ßHC9 cells revealed a possible explanation. As expected, HO activity increased in ßHC9 cells cultured with CoCl2 or CoPP, and the activity was inhibited with ZnPP present during culture. And, although IL-1ß increased HO-1 expression in ßHC9 cells, HO activity was not different from basal, suggesting that an endogenous inhibitor of HO activity may be produced in ß-cells. When HO activity was assessed in homogenates from cells cultured with IL-1ß, a direct stimulatory effect of CoPP on HO was observed. CoCl2 did not mimic the direct effect of CoPP to activate the enzyme, and addition of ZnPP inhibited the HO activity. Thus, a possible explanation for the ability of low levels of CoPP to protect the ß-cells from the effects of IL-1ß is that CoPP directly activated HO activity, even when expression levels of HO were relatively low. In addition, CoPP activation of HO-2 in islets may contribute to increased insulin release.
In summary, these results suggest that the induction and/or activation of HO, in response to Co2+, CoPP, or hemin in islet cells, provides a protective mechanism against the actions of IL-1ß and preserves the glucose-stimulated insulin secretory response and glucose metabolism. The mechanism by which CoCl2 induces HO-1 expression is not known, although studies have described effects of oxidant stress (54), CoPP generation (51), and activation of distal response elements in HO-1 gene regulation (10) as mediating Co2+ responses. Moreover, the results indicate that inhibition of iNOS activity, perhaps mediated through the HO-1-generated product carbon monoxide, plays a role in the protective effect of Co2+ on cytokine-treated islet cells. It is also possible that the other products of HO activity, the antioxidant bilirubin (1), or the iron produced that can consume NO (55), have a protective role in the actions of Co2+ and HO-1 expression.
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Acknowledgments |
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Footnotes |
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2 Portions of this research were completed in partial fulfillment of
the degree of Doctor of Philosophy.
Received April 6, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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via an L-arginine-dependent nitric oxide generating
mechanism. FEBS Lett 276:42–44[CrossRef][Medline]
B in the signaling mechanism. Endocrinology 136:4790–4795[Abstract]
-cells.
Immunol Lett 48:145–148[CrossRef][Medline]
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 N. R. Barshes, S. Wyllie, and J. A. Goss Inflammation-mediated dysfunction and apoptosis in pancreatic islet transplantation: implications for intrahepatic grafts J. Leukoc. Biol., May 1, 2005; 77(5): 587 - 597. [Abstract] [Full Text] [PDF]
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 H. Mosen, A. Salehi, P. Alm, R. Henningsson, J. Jimenez-Feltstrom, C.-G. Ostenson, S. Efendic, and I. Lundquist Defective Glucose-Stimulated Insulin Release in the Diabetic Goto-Kakizaki (GK) Rat Coincides with Reduced Activity of the Islet Carbon Monoxide Signaling Pathway Endocrinology, March 1, 2005; 146(3): 1553 - 1558. [Abstract] [Full Text] [PDF]
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I. Lundquist, P. Alm, A. Salehi, R. Henningsson, E. Grapengiesser, and B. Hellman Carbon monoxide stimulates insulin release and propagates Ca2+ signals between pancreatic {beta}-cells Am J Physiol Endocrinol Metab, November 1, 2003; 285(5): E1055 - E1063. [Abstract] [Full Text] [PDF]
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 L. Gunther, P. O. Berberat, M. Haga, S. Brouard, R. N. Smith, M. P. Soares, F. H. Bach, and E. Tobiasch Carbon Monoxide Protects Pancreatic {beta}-Cells From Apoptosis and Improves Islet Function/Survival After Transplantation Diabetes, April 1, 2002; 51(4): 994 - 999. [Abstract] [Full Text] [PDF]
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D. R. Laybutt, H. Kaneto, W. Hasenkamp, S. Grey, J.-C. Jonas, D. C. Sgroi, A. Groff, C. Ferran, S. Bonner-Weir, A. Sharma, et al. Increased Expression of Antioxidant and Antiapoptotic Genes in Islets That May Contribute to {beta}-Cell Survival During Chronic Hyperglycemia Diabetes, February 1, 2002; 51(2): 413 - 423. [Abstract] [Full Text] [PDF]
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A. Pileggi, R. D. Molano, T. Berney, P. Cattan, C. Vizzardelli, R. Oliver, C. Fraker, C. Ricordi, R. L. Pastori, F. H. Bach, et al. Heme Oxygenase-1 Induction in Islet Cells Results in Protection From Apoptosis and Improved In Vivo Function After Transplantation Diabetes, September 1, 2001; 50(9): 1983 - 1991. [Abstract] [Full Text] [PDF]
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 R. Henningsson, P. Alm, and I. Lundquist Evaluation of islet heme oxygenase-CO and nitric oxide synthase-NO pathways during acute endotoxemia Am J Physiol Cell Physiol, May 1, 2001; 280(5): C1242 - C1254. [Abstract] [Full Text] [PDF]
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 P. Navarra, F. Miceli, G. Tringali, F. Minici, M. G. Pardo, A. Lanzone, S. Mancuso, and R. Apa Evidence for a Functional Link between the Heme Oxygenase-Carbon Monoxide Pathway and Corticotropin-Releasing Hormone Release from Primary Cultures of Human Trophoblast Cells J. Clin. Endocrinol. Metab., January 1, 2001; 86(1): 317 - 323. [Abstract] [Full Text]
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 T. Polte, A. Abate, P. A. Dennery, and H. Schroder Heme Oxygenase-1 Is a cGMP-Inducible Endothelial Protein and Mediates the Cytoprotective Action of Nitric Oxide Arterioscler Thromb Vasc Biol, May 1, 2000; 20(5): 1209 - 1215. [Abstract] [Full Text] [PDF]
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